Use of multiple transformation enhancer sequences to improve plant transformation efficiency

ABSTRACT

The present invention relates to methods and compositions for improving the efficiency of  Agrobacterium - and  Rhizobium -mediated plant cell transformation by use of additional transformation enhancer sequences, such as overdrive or TSS sequences, operably linked to a T-DNA border sequence on a recombinant construct that comprises T-DNA.

This application claims the priority of U.S. Provisional PatentApplication 60/831,814, filed Jul. 19, 2006, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to plant biotechnology. Morespecifically, the invention relates to methods and compositions forimproving the efficiency of bacterially-mediated plant transformation.

2. Description of Related Art

During natural Agrobacterium-mediated transformation of plant cells, apiece of DNA from the Ti plasmid of A. tumefaciens or Ri plasmid of A.rhizogenes is transferred into the plant cell (e.g. Gelvin, 2003). Thistransferred DNA (T-DNA) fragment is flanked by imperfect 24 bp directrepeats that are recognized by Agrobacterium endonuclease VirD2 toproduce a single stranded T-strand by nicking at a specific site in onestrand of each repeat. The repeat that initiates formation of singlestranded T-strand has been termed the “right border” (RB), while therepeat terminating formation of single-stranded T-DNA has been termedthe “left border” (LB). The VirD2 protein is attached to the 5′ end ofthe strand after nicking, and guides the T-strand into plant cells wherethe T-strand is integrated into the plant genome with the help of otherAgrobacterium and plant-encoded proteins. Sequences downstream (in a 5′to 3′ direction) of the T-DNA region, including vector backbonesequence, may be transferred as well (e.g. Kononov et al., 1997). Thislikely occurs by inefficient nicking of at least one of the borders inAgrobacterium prior to transfer to a plant cell.

Comparison of the RB and LB sequences from a variety of Agrobacteriumstrains indicated that both RB and LB share a consensus motif (Canadayet al., 1992), which indicates that other elements may be involved inmodulating the efficiency of RB processing. Cis-acting sequences next tothe RB are present in many Agrobacterium strains, including A.tumefaciens and A. rhizogenes. These sequences are necessary for wildtype virulence (Veluthambi et al., 1988; Shurvington and Ream, 1991;Toro et al., 1989; Toro et al., 1988; Hansen et al., 1992). The sequencein A. tumefaciens was called an “overdrive” or “T-DNA transmissionenhancer” by Peralta et al., (1986). In A. rhizogenes the sequence hasbeen termed the “T-DNA transfer stimulator sequence” (TSS) by Hansen etal (1992). The overdrive (“OD”) sequence was initially defined as aparticular 24 bp motif present immediately in front of the RB repeat ofoctopine Ti TL-DNA (Peralta et al., 1986). A similar sequence is presentin front of the RB repeat of octopine Ti TR-DNA and also in front ofnopaline Ti RB and agropine Ri TL right border (Peralta et al., 1986,Shaw et al., 1984, Barker et al., 1983, Slighton et al., 1985). Furthercomparison of different A. tumefaciens strains revealed a 8 bp overdrivecore sequence present in front of all right border sequences includingnopaline strain pTiT37, octopine strain pTiA6 and A. rhizogenes pRiA4(Peralta et al., 1986).

The presence of octopine overdrive sequence enhanced single strand T-DNAformation in Agrobacterium cells and improved T-DNA transfer into plantcells, and was necessary for wild type virulence (Peralta et al., 1986,Shurvinton and Ream 1991). The LB repeat from nopaline-producing Tiplasmid pTiT37 is capable of producing single-stranded T-DNA with highefficiency when the pTiT37 RB proximal cis-acting sequence was placed infront of it, indicating that an overdrive-like sequence indeed is alsopresent on a nopaline Ti plasmid (Culianez-Macia and Hepburn 1988,Peralta et al., 1986), just as it is in the other identified(octopine-producing) Ti plasmids. Integration of a heterologous octopineoverdrive sequence in front of nopaline pTiT37 RB resulted in muchgreater virulence than the parental strain which contained only asynthetic pTiT37 RB repeat (Peralta et al., 1986).

The VirC1 protein binds to overdrive and is thought to improve VirD2nicking (Toro et al., 1988, 1989), while mutation of virC results inattenuated virulence in plants (Close et al., 1987) and reducedproduction of processed single stranded T-DNA sequence. Both A.tumefaciens octopine and nopaline Ti plasmids contain virC and cancomplement the virC mutation in trans to restore the attenuatedvirulence to wild type level (Close et al., 1987).

The TSS found in A. rhizogenes strains 8196, A4 and 2659 plays a similarrole as the overdrive sequence in A. tumefaciens. Each A. rhizogenesstrain has a different but related sequence (Hansen et al., 1992). The 8bp TSS core sequence repeats 5 times in pRiA4, 6 times in pRi8196 and 17times (rather than 16× as Hansen et al., 1992) in pRi2659 (Genbankaccession AJ271050). pRiA4 has a conserved 8 bp overdrive core sequencein addition to the repeats. Shorter core sequence repeats in pRiA4 andpRi8196 were not sufficient for wild type virulence (Hansen et al.,1992).

Depicker et al. (U.S. Patent Publication 2003/0140376, and correspondinginternational publication WO01/44482) describe recombinant constructswith modified T-DNA borders in order to lessen or prevent transferenceof vector backbone sequences. Conner et al., (WO 05/121346) describecreation and use of sequences from T-DNA border-like regions thatcomprise sequences derived from plants. Heim et al. (U.S. Publ.2003/0188345) describe vectors for Agrobacterium-mediated transformationof plants with modified border regions. Lassner et al., (U.S. Publ.2006/0041956) describe modifications to T-DNA border regions to enableidentification of transgenic events that do not comprise non T-DNAsequences.

While the foregoing studies have increased understanding in the art,what remains needed is a method to improve the efficiency ofAgrobacterium-mediated plant transformation. Although the presence ofoverdrive or TSS sequences increases virulence of wild typeAgrobacterium and improves T-DNA transfer into plant cells compared toplasmids lacking the sequences, it has remained unclear how to furtherimprove transformation efficiency including through the use of overdriveor TSS sequences.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of increasing theefficiency of bacterially-mediated plant transformation, comprising thesteps of: a) introducing at least one additional transformation enhancersequence into a plant transformation vector comprising at least oneT-DNA border region; and b) transforming a plant cell with the vector bybacterially-mediated transformation, wherein the bacterium is competentfor the transformation of the plant cell. The method may optionallycomprise regenerating a transgenic plant from the plant cell. In oneembodiment, the additional transformation enhancer sequence comprises aconsensus core sequence of TGTWTGTK (SEQ ID NO:20). In otherembodiments, the additional transformation enhancer sequence is selectedfrom the group consisting of: SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQID NO:9, SEQ ID NO:13, and a sequence complementary to any of SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:13. Inparticular embodiments, the invention provides a recombinant DNAconstruct comprising SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.

The transformation enhancer sequence used with the invention may belocated proximal to a T-DNA border region or sequence, such as a rightborder (RB) sequence, i.e. between flanking sequence such as vectorsequence and the border sequence. The transformation enhancer sequencemay be from a Ti plasmid of A. tumefaciens, such as a nopaline oroctopine plasmid, or may be from an Ri plasmid of A. rhizogenes. Incertain embodiments, the bacterially-mediated transformation may utilizea technique selected from Agrobacterium-mediated transformation,Rhizobium-mediated transformation, and Sinorhizobium-, Mesorhizobium- orBradyrhizobium-mediated transformation. In certain embodiments, thetransformation enhancer sequence may comprise SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:17, or SEQ ID NO:18. In further embodiments, the T-DNAborder region may comprise from 1 to about 18 copies of thetransformation enhancer sequence, including from about 2 or about 4 toabout 18 copies of the transformation enhancer sequence.

A plant cell in accordance with the invention may be any plant cell. Incertain embodiments, the plant cell is from a plant selected from thegroup consisting of soybean, corn, cotton, canola, rice, wheat, alfalfa,common bean, peanut, tobacco, sunflower, barley, beet, broccoli,cabbage, carrot, cauliflower, celery, Chinese cabbage, cucumber,eggplant, leek, lettuce, melon, oat, onion, pea, pepper, peanut, potato,pumpkin, radish, sorghum, spinach, squash, sugarbeet, tomato andwatermelon. In particular embodiments, the plant cell is a corn cell ora soybean cell.

In another aspect, the invention provides a recombinant DNA constructcomprising a T-DNA border sequence of a Ti or Ri plasmid, operablylinked to a transformation enhancer sequence that comprises two or morecopies of a sequence selected from the group consisting of: SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13, a sequencecomplementary to any of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, or SEQ ID NO:13, and combinations thereof. In particularembodiments, the invention provides a recombinant DNA constructcomprising SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.

In such a construct, the enhancer sequence may comprise at least aboutfour copies of the sequence. The border sequence may be a right border(RB) or left border (LB) sequence. In certain embodiments, the constructmay comprise SEQ ID NO:10 and/or SEQ ID NO:11. The RB sequence may befrom a nopaline Ti plasmid, or an agropine, mannopine, succimanopine,cucumopine, or octopine Ti or Ri plasmid and may comprise SEQ ID NO:12.

In another aspect, the invention provides a cell transformed with aconstruct provided herein. The cell may be a plant or bacterial cell,including an Agrobacterium cell and Rhizobium cell. In one embodiment,the plant cell is from a plant selected from the group consisting ofsoybean, corn, cotton, canola, rice, wheat, alfalfa, common bean,peanut, tobacco and sunflower. The invention also provides transgenicplants transformed a construct of the invention. In particularembodiments, the transgenic plant may be selected from the groupconsisting of soybean, corn, cotton, canola, rice, wheat, alfalfa,common bean, peanut, tobacco and sunflower.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to thedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1: Outline of various transformation enhancer sequences used forimproving transformation efficiency.

FIG. 2: Schematic map of pMON87464.

FIG. 3: Schematic map of pMON87465.

FIG. 4: Engineered RB sequences; overdrive sequence is in bold and the24 bp RB core sequence underlined. (A) sequence of the Nopaline RB+1×overdrive (SEQ ID NO:14); (B) Nopaline RB+4× overdrive (SEQ ID NO:15);(C) Nopaline RB+18× TSS (SEQ ID NO:16).

DESCRIPTION OF SEQUENCE LISTING SEQ ID NO: 1 Forward primer Xd463 for 2XOD sequence preparation. SEQ ID NO: 2 Reverse primer Xd464 for 2X ODsequence preparation. SEQ ID NO: 3 Forward primer Xd465 for 6X TSSsequence preparation. SEQ ID NO: 4 Reverse primer Xd466 for 6X TSSsequence preparation. SEQ ID NO: 5 24 bp core OD of pTiA6. SEQ ID NO: 68 bp 1x TSS sequence. SEQ ID NO: 7 30 bp 1x OD of pTiA6; reversecomplement of SEQ ID NO: 17. SEQ ID NO: 8 1x OD sequence from pTiAB3.SEQ ID NO: 9 1x OD from pTi15955. SEQ ID NO: 10 4X stacked OD. SEQ IDNO: 11 18X stacked TSS. SEQ ID NO: 12 Border region with 1X OD sequence.SEQ ID NO: 13 Partial OD sequence. SEQ ID NO: 14 Nopaline RB region with1X OD. SEQ ID NO: 15 Nopaline RB region with 4X OD. SEQ ID NO: 16Nopaline RB region with 18X TSS. SEQ ID NO: 17 1X OD; reverse complementof SEQ ID NO: 7. SEQ ID NO: 18 4X stacked OD; reverse complement of SEQID NO: 10. SEQ ID NO: 19 Consensus OD sequence (Toro et al., 1988). SEQID NO: 20 Consensus 8 bp core OD sequence.

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of the invention provided to aidthose skilled in the art in practicing the present invention. Those ofordinary skill in the art may make modifications and variations in theembodiments described herein without departing from the spirit or scopeof the present invention.

The invention provides methods and compositions for improving theefficiency of Agrobacterium-mediated transformation of plant cells.Sequencing of the 20 kb T-DNA region of A. rhizogenes K599, a soybeansuper virulent strain, led to the recognition that the pRi plasmid in A.rhizogenes K599 is identical to the A. rhizogenes NCPBB 2659 strain. Thesuper-virulence of the K599 strain may thus be related to the number ofTSS sequences present near the RB. Therefore, stacking of multipleoverdrive and TSS repeats was tested in binary vectors with a nopalineRB (e.g. from pTiT37) to improve transformation efficiency. The octopineTi plasmid's 30 bp overdrive (Shurvinton and Ream 1991) from pTiA6,present in 4 copies, and the A. rhizogenes NCPBB2659 TSS 8 bp coresequence, present in 18 copies, was used to enhance T-DNA transmissionefficiency.

Transformation studies comparing the use of constructs containingvarying numbers of overdrive or TSS sequences demonstrated that thepresence of additional “stacked” copies of these sequences improvedtransformation efficiency by improving transformation frequency as wellas the quality of the resulting transgenic events. For example, theproportion of events with single copy insertions, and also lackingvector backbone sequences (e.g. oriV), was increased. Increasedtransformation frequency and quality events improve the overallefficiency of the transformation process by reducing the amount ofresources required to select event for further commercial development.

The invention therefore provides improved methods for obtaining fertiletransgenic plants and for the transformation of plant cells or tissuesand regeneration of the transformed cells or tissues into fertiletransgenic plants. To initiate a transformation process in accordancewith the invention, the genetic components desired to be inserted intothe plant cells or tissues will first be selected. Genetic componentsmay include any nucleic acid that is to be introduced into a plant cellor tissue using the method according to the invention. Geneticcomponents can include non-plant DNA, plant DNA, or synthetic DNA.

In certain embodiments of the invention, genetic components areincorporated into a DNA composition such as a recombinant,double-stranded plasmid or vector molecule comprising genetic componentssuch as: (a) a promoter that functions in plant cells to cause theproduction of an RNA sequence, (b) a structural DNA sequence that causesthe production of an RNA sequence that encodes a desired protein orpolypeptide, and (c) a 3′ non-translated DNA sequence that functions inplant cells to cause the polyadenylation of the 3′ end of the RNAsequence. The vector may also contain genetic components that facilitatetransformation and regulate expression of the desired gene(s).

The genetic components are typically oriented so as to express an mRNA,which in one embodiment can be translated into a protein. The expressionof a plant structural coding sequence (a gene, cDNA, synthetic DNA, orother DNA) that exists in double-stranded form involves transcription ofmessenger RNA (mRNA) from one strand of the DNA by RNA polymerase andsubsequent processing of the mRNA primary transcript inside the nucleus.This processing involves a 3′ non-translated region that includespolyadenylation of the 3′ ends of the mRNA.

General methods for preparing plasmids or vectors that contain desiredgenetic components and can be used to transform plants and methods ofmaking those vectors are known in the art. Vectors typically consist ofa number of genetic components, including, but not limited to,regulatory elements such as promoters, leaders, introns, and terminatorsequences. Regulatory elements are also referred to as cis- ortrans-regulatory elements, depending on the proximity of the element tothe sequences or gene(s) they control. The promoter region contains asequence of bases that signals RNA polymerase to associate with the DNAand to initiate the transcription into mRNA using one of the DNA strandsas a template to make a corresponding complementary strand of RNA.

The constructs may also contain the plasmid backbone DNA segments thatprovide replication function and antibiotic selection in bacterialcells, for example, an Escherichia coli origin of replication such asori322, a broad host range origin of replication such as oriV or oriRi,and a coding region for a selectable marker such as Spec/Strp thatencodes for Tn7 aminoglycoside adenyltransferase (aadA) conferringresistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent)selectable marker gene. For plant transformation, the host bacterialstrain is often Agrobacterium tumefaciens ABI, C58, LBA4404, EHA101, orEHA105 carrying a plasmid having a transfer function for the expressionunit. Other bacterial strains known to those skilled in the art of planttransformation can function in the present invention, including A.rhizogenes, Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp.,and Rhizobium sp. strains.

A number of promoters that are active in plant cells have been describedin the literature. Such promoters include, but are not limited to, thenopaline synthase (NOS) and octopine synthase (OCS) promoters, which arecarried on tumor-inducing plasmids of A. tumefaciens; the caulimoviruspromoters such as the cauliflower mosaic virus (CaMV) 19S and 35Spromoters and the figwort mosaic virus (FMV) 35S promoter; the enhancedCaMV35S promoter (e35S); and the light-inducible promoter from the smallsubunit of ribulose bisphosphate carboxylase (ssRUBISCO, a very abundantplant polypeptide). All of these promoters have been used to createvarious types of DNA constructs that have been expressed in plants.Promoter hybrids can also be constructed to enhance transcriptionalactivity or to combine desired transcriptional activity, inducibility,and tissue or developmental specificity.

Thus, promoters that function in plants may be inducible, viral,synthetic, constitutive as described, temporally regulated, spatiallyregulated, and/or spatio-temporally regulated. Other promoters that aretissue-enhanced, tissue-specific, or developmentally regulated are alsoknown in the art and envisioned to have utility in the practice of thisinvention. Useful promoters may be obtained from a variety of sourcessuch as plants and plant DNA viruses. It is preferred that theparticular promoter selected should be capable of causing sufficientexpression to result in the production of an effective amount of thegene product of interest.

The promoters used in the DNA constructs (i.e., chimeric/recombinantplant genes) of the present invention may be modified, if desired, toaffect their control characteristics. Promoters can be derived by meansof ligation with operator regions, random or controlled mutagenesis,etc. Furthermore, the promoters may be altered to contain multiple“enhancer sequences” to assist in elevating gene expression.

The mRNA produced by a DNA construct of the present invention may alsocontain a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNAs, fromsuitable eukaryotic genes, or from a synthetic gene sequence. Such“enhancer” sequences may be desirable to increase or alter thetranslational efficiency of the resultant mRNA and are usually known astranslational enhancers. Other genetic components that serve to enhanceexpression or affect transcription or translation of a gene are alsoenvisioned as genetic components. The 3′ non-translated region of thechimeric constructs preferably contains a transcriptional terminator, oran element having equivalent function, and a polyadenylation signal,which functions in plants to polyadenylate the 3′ end of the RNA.Examples of suitable 3′ regions are (1) the 3′ transcribed,non-translated regions containing the polyadenylation signal ofAgrobacterium tumor-inducing (Ti) plasmid genes, such as the nopalinesynthase (nos) gene, and (2) plant genes such as the soybean storageprotein genes and the small subunit of the ribulose-1,5-bisphosphatecarboxylase (ssRUBISCO) gene. An example of a preferred 3′ region isthat from the ssRUBISCO E9 gene from pea.

Typically, DNA sequences located a few hundred base pairs downstream ofthe polyadenylation site serve to terminate transcription. These DNAsequences are referred to herein as transcription-termination regions.The regions are required for efficient polyadenylation of transcribedmessenger RNA (mRNA) and are known as 3′ non-translated regions. RNApolymerase transcribes a coding DNA sequence through a site wherepolyadenylation occurs.

In many transformation systems, it is preferable that the transformationvector contains a selectable, screenable, or scoreable marker gene.These genetic components are also referred to herein as functionalgenetic components, as they produce a product that serves a function inthe identification of a transformed plant, or a product of desiredutility.

The DNA that serves as a selection device may function in a regenerableplant tissue to produce a compound that confers upon the plant tissueresistance to an otherwise toxic compound. Genes of interest for use asa selectable, screenable, or scoreable marker would include, but are notlimited to, β-glucuronidase (gus), green fluorescent protein (gfp),luciferase (lux), antibiotics like kanamycin (Dekeyser et al., 1989),genes allowing tolerance to herbicides like glyphosate (Della-Cioppa etal., 1987), such as CP4 EPSPS (U.S. Pat. No. 5,627,061; U.S. Pat. No.5,633,435; U.S. Pat. No. 6,040,497; U.S. Pat. No. 5,094,945;WO04/074443; WO04/009761); glufosinate (U.S. Pat. No. 5,646,024, U.S.Pat. No. 5,561,236, U.S. Pat. No. 5,276,268; U.S. Pat. No. 5,637,489;U.S. Pat. No. 5,273,894); 2,4-D (WO05/107437) and dicamba, such as DMO(U.S. Pat. No. 7,022,896). Other selection methods can also beimplemented, including, but not limited to, tolerance tophosphinothricin, bialaphos, and positive selection mechanisms (Joersboet al., 1998) and would still fall within the scope of the presentinvention. Examples of various selectable/screenable/scoreable markersand genes encoding them are disclosed in Miki and McHugh (2004).

The present invention can be used with any suitable plant transformationplasmid or vector containing a selectable or screenable marker andassociated regulatory elements as described, along with one or morenucleic acids (a structural gene of interest) expressed in a mannersufficient to confer a particular desirable trait. Examples of suitablestructural genes of interest envisioned by the present inventioninclude, but are not limited to, genes for insect or pest tolerance,genes for herbicide tolerance, genes for quality improvements such asyield, nutritional enhancements, environmental or stress tolerances, orgenes for any desirable changes in plant physiology, growth,development, morphology, or plant product(s).

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viadouble-stranded RNA mediated mechanisms, including antisense- andcosuppression-mediated mechanisms (see, for example, Bird et al., 1991).The RNA could also be a catalytic RNA molecule (i.e., a ribozyme)engineered to cleave a desired endogenous mRNA product (see for example,Gibson and Shillitoe, 1997). More particularly, for a description ofantisense regulation of gene expression in plant cells see U.S. Pat. No.5,107,065, and for a description of gene suppression in plants bytranscription of a dsRNA see U.S. Pat. No. 6,506,559, U.S. PatentApplication Publication No. 2002/0168707 A1, and U.S. patent applicationSer. No. 09/423,143 (see WO 98/53083), Ser. No. 09/127,735 (see WO99/53050) and Ser. No. 09/084,942 (see WO 99/61631), all of which areincorporated in their entirety herein by reference.

Use of sequences that result in silencing of other endogenous genes(e.g. RNAi technologies including miRNA) to result in a phenotype isalso envisioned. For instance RNAi may be used to silence one or moregenes resulting in a scoreable phenotype. One embodiment is to assemblea DNA cassette that will transcribe an inverted repeat of sequences, toproduce a double-stranded RNA (dsRNA), typically at least about 19-21 bpin length and corresponding to a portion of one or more genes targetedfor silencing. Thus, any gene that produces a protein or mRNA thatexpresses a phenotype or morphology change of interest is useful for thepractice of the present invention.

Exemplary nucleic acids that may be introduced by the methodsencompassed by the present invention include, for example, heterologousDNA sequences—that is, sequences or genes from another species, or evengenes or sequences that originate with or are present in the samespecies but are incorporated into recipient cells by genetic engineeringmethods rather than classical reproduction or breeding techniques. Theterm heterologous, however, is also intended to refer to genes that arenot normally present in the cell being transformed or to genes that arenot present in the form, structure, etc., as found in the transformingDNA segment or to genes that are normally present but a differentexpression is desirable. Thus, the term “heterologous” gene or DNA isintended to refer to any gene or DNA segment that is introduced into arecipient cell, regardless of whether a similar gene may already bepresent in such a cell. The type of DNA included in the heterologous DNAcan include DNA that is already present in the plant cell, DNA fromanother plant, DNA from a different organism, or a DNA generatedexternally, such as a DNA sequence containing an antisense message of agene, or a DNA sequence encoding a synthetic or modified version of agene or sequence.

In light of this disclosure, numerous other possible selectable orscreenable marker genes, regulatory elements, and other sequences ofinterest will be apparent to those of skill in the art. Therefore, theforegoing discussion is intended to be exemplary rather than exhaustive.

After the construction of the plant transformation vector or construct,the nucleic acid molecule, prepared as a DNA composition in vitro, isgenerally introduced into a suitable host such as Escherichia coli andmated into another suitable host such as Agrobacterium or Rhizobium, ordirectly transformed into competent Agrobacterium or Rhizobium. Thesetechniques are well-known to those of skill in the art and have beendescribed for a number of plant systems including soybean, cotton, andwheat (see, for example, U.S. Pat. Nos. 5,569,834 and 5,159,135 and WO97/48814). Those of skill in the art would recognize the utility ofAgrobacterium-mediated transformation methods. Strains may include, butare not limited to, disarmed derivatives of A. tumefaciens strain C58, anopaline strain that is used to mediate the transfer of DNA into a plantcell; octopine strains, such as LBA4404; or agropine strains, e.g.,EHA101, EHA105, or R. leguminosarum USDA2370 with a Ti or Ri plasmid.The use of these strains for plant transformation has been reported, andthe methods are familiar to those of skill in the art.

Plant tissue to be transformed is typically inoculated and co-culturedwith Agrobacterium or Rhizobium containing a recombinant constructcomprising at least one heterologous overdrive or TSS sequence, asequence of interest to be transferred, and at least one RB sequencethat serves to define the DNA to be transferred, and is selected underappropriate conditions. In certain embodiments, at least one LB sequenceis also present on the recombinant construct. In certain otherembodiments, a border sequence can be a “plant derived border-likesequence(s).” Methods of identifying and using such sequences aredescribed in Rommens et al., 2005; Rommens 2004a; Rommens et al., 2004b

The present invention can be used with any transformable cell or tissue.Those of skill in the art recognize that transformable plant tissuegenerally refers to tissue that can have exogenous DNA inserted in itsgenome and under appropriate culture conditions can form into adifferentiated plant. Such tissue can include, but is not limited to,cell suspensions, callus tissue, hypocotyl tissue, cotyledons, embryos,meristematic tissue, roots, and leaves. For example, transformabletissues can include calli or embryoids from anthers, microspores,inflorescences, and leaf tissues. Other tissues are also envisioned tohave utility in the practice of the present invention, and thedesirability of a particular explant for a particular plant species iseither known in the art or may be determined by routine screening andtesting experiments whereby various explants are used in thetransformation process and those that are more successful in producingtransgenic plants are identified.

Methods for transforming dicots by use of Agrobacterium or Rhizobium andobtaining transgenic plants have been published for a number of cropsincluding cotton, soybean, Brassica, and peanut. Successfultransformation of monocotyledonous plants by Agrobacterium- orRhizobium-based methods has also been reported. Transformation and plantregeneration have been achieved and reported at least in asparagus,barley, maize, oat, rice, sugarcane, tall fescue, and wheat. Techniquesthat may be particularly useful in the context of cotton transformationare disclosed in U.S. Pat. Nos. 5,846,797, 5,159,135, 5,004,863, and6,624,344. Techniques for transforming Brassica plants in particular aredisclosed, for example, in U.S. Pat. No. 5,750,871. Techniques fortransforming soybean are disclosed in for example in Zhang et al.,(1999) and U.S. Pat. No. 6,384,301; and techniques for transforming cornare disclosed in for example in U.S. Pat. No. 5,981,840, U.S. Pat. No.7,060,876, U.S. Pat. No. 5,591,616, WO95/06722, and U.S. Patent Pub.2004/244075.

In one embodiment, after incubation on medium containing antibiotics toinhibit Agrobacterium or Rhizobium growth without selective agents(delay medium), the explants are cultured on selective growth mediumincluding, but not limited to, a callus-inducing medium containing aplant cell selective agent. Typical selective agents have been describedand include, but are not limited to, antibiotics such as G418,paromomycin, kanamycin, or other chemicals such as glyphosate, dicamba,and glufosinate. The plant tissue cultures surviving the selectionmedium are subsequently transferred to a regeneration medium suitablefor the production of transformed plantlets. Regeneration can be carriedout over several steps. Those of skill in the art are aware of thenumerous types of media and transfer requirements that can beimplemented and optimized for each plant system for plant transformationand regeneration.

The transformants produced are subsequently analyzed to determine thepresence or absence of a particular nucleic acid of interest containedon the transformation vector. Molecular analyses can include, but arenot limited to, Southern blots or PCR (polymerase chain reaction)analyses. These and other well known methods can be performed to confirmthe stability of the transformed plants produced by the methodsdisclosed, as well as the copy number of insertions, and the presence ofvector backbone sequences flanking the T-DNA. These methods are wellknown to those of skill in the art and have been reported (see, forexample, Sambrook et al., 1989).

The previous discussion is merely a broad outline of standardtransformation and regeneration protocols. One of skill in the art knowsthat specific crops and specific protocols can vary somewhat from thebroad outline. A variety of media can be used in each system as well.Those of skill in the art are familiar with the variety of tissueculture media that, when supplemented appropriately, support planttissue growth and development. These tissue culture media can either bepurchased as a commercial preparation or custom prepared and modified bythose of skill in the art. Examples of such media include, but are notlimited to those described by Murashige and Skoog (1962); Chu et al.(1975); Linsmaier and Skoog (1965); Uchimiya and Murashige (1962);Gamborg et al. (1968); Duncan et al. (1985); McCown and Lloyd (1981);Nitsch and Nitsch (1969); and Schenk and Hildebrandt (1972), orderivations of these media supplemented accordingly. Those of skill inthe art are aware that media and media supplements such as nutrients andgrowth regulators for use in transformation and regeneration are usuallyoptimized for the particular target crop or variety of interest.Reagents are commercially available and can be purchased from a numberof suppliers (see, for example Sigma Chemical Co., St. Louis, Mo.).

“Overdrive” sequences have been identified in numerous Ti plasmids,including pTiA6, pTiAB3, and pTi15955. Other sequences with highsimilarity to overdrive or TSS can be identified, for example, using the“BestFit,” “Gap,” or “FASTA” programs of the Sequence Analysis SoftwarePackage, Genetics Computer Group, Inc., University of WisconsinBiotechnology Center, Madison, Wis. 53711, or using the “BLAST” program(Altschul et al., 1990), or another available DNA sequence analysispackage. Such sequences when present in multicopy near an RB sequencemay be assayed for transformation enhancement activity, similarly to thesequences whose enhancer activity is described below.

“Frequency of transformation” or “transformation frequency,” as usedherein, refers to the percentage of transgenic events produced perexplant or the percentage of transgenic plants produced per explant.

“Border sequence,” e.g. right border (RB) or left border (LB), refers toa directly repeated nucleic acid sequence defining an end of thetransferred DNA (T-DNA) region, typically about 24 bp in length. Bordersequences may be from a Ti or Ri plasmid of Agrobacterium sp., or may beplant derived sequences that function similarly.

“T-DNA Border region” refers to the RB or LB sequence and associatedflanking sequence, typically about 100 bp in length, and, as found innature, may include a transformation enhancer sequence.

“Transformation efficiency” as used herein, refers to any improvement,such as increase in transformation frequency and quality events thatimpact overall efficiency of the transformation process by reducing theamount of resources required to select event for further commercialdevelopment.

“Transformation enhancer” as used herein refers to overdrive and TSSsequences.

A first nucleic acid sequence is “operably linked” with a second nucleicacid sequence when the sequences are so arranged that the first nucleicacid sequence affects the function of the second nucleic-acid sequence.Preferably the two sequences are part of a single contiguous nucleicacid molecule. The overdrive or TSS enhancer sequence may be placedimmediately adjacent to the border sequence, such as the RB sequence.Alternatively, in certain embodiments the overdrive or TSS sequence islocated about 1, 10, 25, 50, 100, 250, 500, 1000 or more nucleotidesfrom the end of the border sequence, including all intermediate ranges.The overdrive sequence may be placed in either orientation relative tothe border.

EXAMPLES

Those of skill in the art will appreciate the many advantages of themethods and compositions provided by the present invention. Thefollowing examples are included to demonstrate the preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention. All references cited herein are incorporated herein byreference to the extent that they supplement, explain, provide abackground for, or teach methodology, techniques, or compositionsemployed herein.

Example 1 Synthesis of Transformation Enhancer Sequences

1) Synthesis of 4× Overdrive Sequence

To synthesize a 4× 30 bp overdrive (OD) sequence (5′caaacaaacatacacagcgacttattcacacaaacaaacatacacagcgacttattcacacaaacaaacatacacagcgacttattcacacaaacaaacatacacagcgacttattcaca 3′; SEQ ID NO:18), 2× 30 bp overdriveprimer pair 5′caaacaaacatacacagcgacttattcacacaaacaaacatacacagcgacttattcaca 3′ (Xd463;SEQ ID NO:1) and 5′tgtgaataagtcgctgtgtatgtttgtttgtgtgaataagtcgctgtgtatgtttgtttg 3′ (Xd464;SEQ ID NO:2) were synthesized, mixed and PCR amplified for 20 cycles inthe presence of high fidelity PfuTurbo® polymerase from Stratagene (LaJolla, Calif.). The PCR product was fractionated on a 1% Agarose gel,and the portion of the gel corresponding to the size ranging between100-300 bp was excised, purified and ligated into TOPO Zero blunt PCRvector from Invitrogen (Carlsbad, Calif.). The repeat stacking wasconfirmed by sequencing. Up to 6× overdrive sequence was observedfollowing PCR, although only 4× 30 bp overdrive insert was utilized insubsequent cloning of a multicopy overdrive construct.

2) Synthesis of 18× TSS Sequence

6× 8 bp TSS repeat primer pairs: 5′ctgacgaactgacgaactgacgaactgacgaactgacgaactgacgaa 3′ (Xd465; SEQ IDNO:3), and 5′ ttcgtcagttcgtcagttcgtcagttcgtcagttcgtcagttcgtcag 3′(Xd466; SEQ ID NO:4) were synthesized and equally mixed and amplifiedfor 5 cycles in the presence of Pfu Turbo® polymerase from Stratagene(La Jolla, Calif.). The 100-300 bp size gel slice was cut, purified andligated into TOPO Zero blunt PCR vector from Invitrogen. Up to 35× TSSrepeat was confirmed by sequencing, but only 18× TSS repeat was kept forfurther cloning. The size of overdrive and TSS was dependent on the PCRcycles and the excised gel position.

Example 2 Construction of Vectors Having RB with Overdrive, AdditionalOverdrive, or 18× TSS

To place the overdrive or TSS in front of a 24 bp RB, an EcoRI site wasintroduced into a nopaline RB, 11 bp away from the upstream of the RB(of pMON83900). The 4× overdrive or 18× TSS was excised from thecorresponding TOPO cloning vector digested by EcoRI and inserted intopMON83900 opened by EcoRI, resulting in pMON83903 and pMON83909,respectively.

The modified RB containing either 4× overdrive or the 18× TSS frompMON83903 or pMON83909 were digested with HindIII/SpeI and used toreplace the 1× overdrive RB of pMON83902 with the HindIII/SpeI fragmentcomprising the 4× overdrive or 18× TSS enhancer sequences, resulting inpMON87462 and pMON83864, respectively, for soy transformation.Alternatively, the RB of pMON80105 was modified so as to comprise the 4×overdrive or 18× TSS by inserting the SpeI/SalI fragment from pMON83903or pMON83909 to yield pMON87465 and pMON87466, respectively, for corntransformation. The modified RB with 1×, 4× and 18× transformationenhancer sequences are shown in FIGS. 1 and 4 and SEQ ID NOs:14-16.

A construct containing 1× overdrive sequence (SEQ ID NO:17) wassynthesized by first assembling the oligonucleotide containing the 30 bpoverdrive sequence according to standard protocol and then cloning itinto pBlueScript®II (Stratagene Inc., La Jolla, Calif.), resulting inpMON80088. Then the SpeI and NotI (filled-in with polymerase) fragmentfrom pMON80088 was inserted into pMON80105 digested with SpeI and SmaI,resulting into pMON80121 for corn transformation. For soybeantransformation, 1× overdrive RB construct, pMON83902, was made byreplacing the RB in pMON83898 with the 1× overdrive RB fragment frompMON80121 using PmeI/NdeI restriction enzyme sites.

Example 3 Transformation of Corn with Overdrive or TSS-Enhanced RBSequences

Corn (Zea mays) cells were transformed with oriV containing vectorspMON80105, pMON80121, pMON87465, or pMON87466 essentially as describedin U.S. Patent Application Publn. 2004/244075 in order to assess theability of stacking of additional overdrive and TSS enhancer sequencesto improve transformation frequency and the proportion of eventscomprising low copy number T-DNA insertion and lacking vector backbonesequence (e.g. E. coli-derived oriV). The control treatment consisted oftransformation with pMON80105, lacking an overdrive or TSS sequence. Asshown in Table 1, use of constructs comprising stacked enhancersequences resulted in a statistically significant increase intransformation frequency. With these constructs, a higher percentage ofquality TF was also obtained. Quality TF combines TF and events with oneor two copies. Also, the percentage of events having one or two copiesincreased.

TABLE 1 Effect of transformation enhancer sequences on transformationfrequency and event quality in corn. % of one or two copies eventsOverdrive Construct % Transformation % Quality regardless of in RB(pMON) Frequency (TF)^(a) TF backbone 4X OD 87465 25.3 12.7% 50.1 1X OD80121 24.1 10.4% 43.2 18X TSS 87466 22.8 10.2% 44.7 Control 80105 17.76.4% 39.0 ^(a)denotes statistical significance

Example 4 Transformation of Soybean with Overdrive or TSS-Enhanced RBSequences

Soybean (Glycine max) cells were transformed with oriV containingvectors pMON83898, pMON83902, pMON87462, or pMON87464 essentially asdescribed in U.S. Pat. No. 6,384,301 in order to assess the ability ofstacking of additional overdrive and TSS enhancer sequences to improvetransformation frequency and the proportion of events comprising lowcopy number T-DNA insertion and lacking vector backbone sequence (e.g.E. coli-derived oriV). The sequences of the stacked 4× overdrive and 18×TSS enhancers are found in SEQ ID NO:10 and SEQ ID NO:11, respectively.The control treatment consisted of transformation with pMON83898,lacking an overdrive or TSS sequence. As shown in Tables 2-3, use ofconstructs comprising stacked transformation enhancer sequences resultedin an increase in transformation frequency. The proportion of singlecopy and backbone free events also increased (Table 3; column 5). Also,the percentage of events having one or two copies increased.

TABLE 2 Effect of transformation enhancer sequences on transformationfrequency in soybean. Transformation Enhancer sequence pMON plasmidfrequency (%) Control 83898 2.79 1X overdrive 83902 3.06 4X overdrive87462 4.22* 18X TSS 87464 4.10* *statistically significant increase

TABLE 3 Effect of transformation enhancer sequences on event quality.oriV oriV 1 or 2 copy positive/ negative/ 1 copy/ regardless of pMONTotal total GOI total GOI oriV oriV presence plasmid events positivepositive negative or absence 83898 31  6/24 18/24 1 19 (control) (25%) (75%)  (4%)   (79%)  83902 61 16/45 29/45 6 30 (1X OD) (35.5%) (64.5%)(13.3%) (66.7%) 87462 43 12/33 21/33 4 23 (4X OD) (36.4%) (63.6%)(12.1%) (69.7%) 87464 71 10/50 40/50 17  37 (18X TSS) (20%)  (80%) (34%)  (74%) 

Example 5 Additional Transformation Enhancer Sequences

In addition to the overdrive sequence of pTiA6 used above (SEQ IDNO:17), other overdrive sequences (including the reverse complementarysequences) are known in the art (e.g. Shurvinton and Ream, 1991), andmay be used similarly. These sequences may include but are not limitedto those from pTiAB3 (GenBank M63056) (TGTGAATAAATCGCTGTGTATGTTTGTTTG;SEQ ID NO:8), and pTi15955 (GenBank AF242881)(TTGTCTAAATTTCTGTATTTGTTTGTTTG; SEQ ID NO:9), and the consensus sequenceAAACAAACATACACAGCGACTTATTCACA (SEQ ID NO:13), andTAARTYNCTGTRTNTGTTTGTTTG; (SEQ ID NO:19, Toro et al., 1988) amongothers. Primers may be synthesized accordingly and PCR carried out asdescribed in Example 1 to create DNA segments comprising these sequencesfor use in construction of recombinant plasmids analogous to pMON83902,pMON80121, pMON87462, and pMON87465, among others. Crop plants can betransformed with constructs comprising one or more of thesetransformation enhancer sequences and can be assessed for their abilityto improve transformation frequency and the proportion of eventscomprising low copy number T-DNA insertion and lacking vector backbonesequence.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method of increasing the efficiency of Rhizobia-mediated planttransformation, comprising: transforming a plant cell with a Rhizobiabacterium, said Rhizobia bacterium comprising a plant transformationvector which comprises at least one T-DNA border sequence and atransformation enhancer sequence operably linked to said T-DNA bordersequence; wherein said transformation enhancer sequence comprises two ormore copies of an overdrive (OD) sequence from Agrobacteriumtumefaciens.
 2. The method of claim 1, wherein the OD sequence comprisesa consensus core sequence of SEQ ID NO:20 or a sequence complementary toSEQ ID NO:20.
 3. The method of claim 2, wherein the OD sequencecomprises a sequence selected from the group consisting of: SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, and a sequence complementary to any of SEQ IDNO:7, SEQ ID NO:8, and SEQ ID NO:9.
 4. The method of claim 1, whereinthe transformation enhancer sequence is located proximal to a T-DNAright border (RB) sequence.
 5. The method of claim 1, wherein the ODsequence is from a nopaline or octopine Ti plasmid of A. tumefaciens. 6.The method of claim 1, wherein the Rhizobia-mediated transformation isAgrobacterium-, Rhizobium-, Sinorhizobium-, Mesorhizobium-, orBradyrhizobium-mediated transformation.
 7. The method of claim 6,wherein the Rhizobia-mediated transformation is Agrobacterium-mediatedtransformation.
 8. The method of claim 1, wherein the plant cell is froma plant selected from the group consisting of soybean, corn, cotton,canola, rice, wheat, alfalfa, common bean, peanut, tobacco, sunflower,barley, beet, broccoli, cabbage, carrot, cauliflower, celery, Chinesecabbage, cucumber, eggplant, leek, lettuce, melon, oat, onion, pea,pepper, peanut, potato, pumpkin, radish, sorghum, spinach, squash,sugarbeet, tomato and watermelon.
 9. The method of claim 1, whereintransformation enhancer sequence comprises SEQ ID NO:10, or a sequencecomplementary to SEQ ID NO:10.
 10. The method of claim 1, wherein theplant cell is a corn or soybean cell.
 11. The method of claim 1, whereinthe transformation enhancer sequence comprises from 2 to about 18 copiesof said OD sequence.
 12. The method of claim 1, further comprising thestep of: c) regenerating a transgenic plant from said plant cell.
 13. Arecombinant DNA construct comprising a T-DNA border sequence operablylinked to a transformation enhancer sequence that comprises two or morecopies of a sequence selected from the group consisting of: SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, a sequence complementary to any of SEQ IDNO:7, SEQ ID NO:8, and SEQ ID NO:9, and combinations thereof.
 14. Theconstruct of claim 13, wherein the enhancer sequence comprises at leastabout four copies of said sequence.
 15. The construct of claim 13,wherein the border sequence is a right border (RB) sequence.
 16. Theconstruct of claim 13, wherein the border sequence is a left border (LB)sequence.
 17. The construct of claim 13, wherein the construct comprisesSEQ ID NO:10.
 18. The construct of claim 15, wherein the RB sequence isfrom a nopaline Ti plasmid.
 19. The construct of claim 15, wherein theRB sequence is from an octopine Ti plasmid.
 20. A transgenic celltransformed with the construct of claim
 13. 21. The cell of claim 20,defined as a plant or bacterial cell.
 22. The cell of claim 21, whereinthe cell is an Agrobacterium cell.
 23. The cell of claim 21, wherein thecell is a Rhizobium cell.
 24. The cell of claim 21, wherein the plantcell is from a plant selected from the group consisting of soybean,corn, cotton, canola, rice, wheat, alfalfa, common bean, peanut,tobacco, sunflower, barley, beet, broccoli, cabbage, carrot,cauliflower, celery, Chinese cabbage, cucumber, eggplant, leek, lettuce,melon, oat, onion, pea, pepper, peanut, potato, pumpkin, radish,sorghum, spinach, squash, sugarbeet, tomato, and watermelon.
 25. Atransgenic plant transformed with the construct of claim
 13. 26. Thetransgenic plant of claim 25, defined as selected from the groupconsisting of soybean, corn, cotton, canola, rice, wheat, alfalfa,common bean, peanut, tobacco, sunflower, barley, beet, broccoli,cabbage, carrot, cauliflower, celery, Chinese cabbage, cucumber,eggplant, leek, lettuce, melon, oat, onion, pea, pepper, peanut, potato,pumpkin, radish, sorghum, spinach, squash, sugarbeet, tomato, andwatermelon.